The Effects Of Gene Recombination On Biological Characteristics Of European Avian-like H1N1 Subtype Swine Influenza Virus

Swine influenza virus(SIV)is segmented,negative-strand RNA virus belonging to the Orthomyxoviridae family.The H1N1 subtype SIVs can be divided into the European avian-like H1N1 subtype SIV,the recombinant H1N1 subtype SIV,the classical H1N1 subtype SIV and the human H1N1 subtype SIV.The European avian-like H1N1 subtype SIV has been rapidly circulating in Europe and Asia since its discovery in Europe in 1979.In recent years,a large number of recombinant H1N1 subtype SIVs have been isolated in swine herds,which seems to indicate that the activity of influenza viruses in pigs is increasingly complicated.However,the effects of gene recombination on the biological characteristics of the European avian-like H1N1 subtype SIV are not yet clear at home and abroad.Based on the molecular epidemiological survey in some regions of China,two representative strains were identified through molecular evolution analysis of SIV,which are the European avian-like H1N1 subtype SIV(A/swine/Tianjin/6/2011(H1N1),TJ6 for short)and the triple-ressortant H1N1 subtype SIV(A/swine/Tianjin /10/2013(H1N1),TJ10 for short).The replication and pathogenicity of two strains were compared in MDCK cells,pigs and mice respectively.The results showed that the replication ability of TJ6 in MDCK cells was lower than TJ10;the effects of TJ10 on weight changes in pigs and mice were more pronounced,resulting in more severe pathological lesions and higher viral titers in the lung and nasal bones.To elucidate the differences in host organism after infection with two H1N1 subtype SIVs,RNA-sequencing technology was used to analyze the discrepancy on mRNA of the lung tissue of mice infected with different viruses.4588 genes were differential expression based on criterions of fold change>=2 or fold change<=1/2 and FDR< 0.01.Compared with the control group,there were 1275 and 3871 differential expression genes in TJ6 and TJ10 group.The group of TJ10 had 2681 differential expression genes in contrast to TJ6 group,of which 1125 and 1556 genes were significantly up-regulated and down-regulated.According to the GO classification statistics,KEGG analysis and eggNOG classification of the differentially expressed genes,a large number of genes in each comparison group were involved in biological processes such as immune response and signal transduction,and also with cancer,influenza A virus,cytokine receptor interaction related pathway.Compared with TJ6,TJ10 triggered a much stronger immune response and inflammatory reaction in the host,causing cytokines expression imblanced severely and immune regulation dysfunction,which lead to the death of mice.In order to further analyze the pathogenic mechanism of virus molecules,the sequences of two strains were determined and the amino acid sequences were analyzed.The results showed that two strains were greatly different in the gene fragments NS,NP,PA,PB1 and PB2.Reverse genetics technology were used to construct the reverse genetic manipulation technology platform of the two viruses respectively,and the rescue strains rTJ6,rTJ10 were obtained.The results showed that the biological characteristics of the rescued strains and the wild strains in MDCK cells and mice were almost the same.A variety of viruses had been rescued and identified through the replacement of single or polygenic fragments.Vitro and in vivo studies showed that the four genes,NP,PA,PB1 and PB2,which were composed of ribonucleoprotein,worked together to increase the activity of polymerase;NS1 promoted the replication of viruses by antagonizing the production of IFN.It was inferred that the virus recombination promoted the transcription and replication of the virus and improved the virus virulence through the synergistic combination of ribonucleoprotein complex genes and NS gene.Gene recombination is the driving force of the evolution of the influenza virus,allowing the influenza virus to acquire antigenic variations that evades the host’s immune response and even triggers the emergence of new pandemic strains.This study helps us to understand the evolutionary pattern of influenza virus and provides a reliable theoretical basis for early warning of influenza pandemic.Moreover,it improves the understanding of the molecular pathogenesis of influenza virus.